In any chemical or bioprocessing industry, the need to separate and purify a product from a complex mixture is a necessary and important step in the production process. Today, there exists a wide market of methods in which industry can accomplish these goals, one of which is chromatography. Chromatography is well suited to a variety of uses in the field of biotechnology since it can separate complex mixtures with great precision; in particular, chromatography is very well suited to the separation of more delicate or sensitive products, such as proteins, since the conditions under which it is performed are not typically severe.
One chromatography method, which is an especially sensitive separation technique and is also applicable to most types of proteins, is metal chelate affinity chromatography (MCAC), also known as immobilised metal ion adsorption chromatography (IMAC). This technique is commonly used in purification schemes together with another chromatographic step, such as ion exchange chromatography (IEX) and/or hydrophobic interaction chromatography (HIC).
More specifically, IMAC utilises matrices that comprise a group capable of forming a chelate with a transition metal ion, which chelate in turn is used as the ligand in chromatography to adsorb a compound from a liquid. The binding strength in IMAC is affected predominately by the species of metal ion, the pH of the buffer and the nature of the ligand used. Since the metal ions are strongly bound to the matrix, the adsorbed protein can be eluted either by lowering the pH or by competitive elution (for example, with imidazole).
In general, IMAC is useful for the separation of proteins or other molecules that present an affinity for the transition metal ion of the matrix. For example, proteins will bind to the matrix upon the presence of accessible histidine, cysteine and tryptophan residues, which all exhibit an affinity for the chelated metal.
With the advent of molecular biological techniques, proteins are now easily tailored or tagged with one or more histidine residues in order to increase their affinity to metal chelated ligands, and accordingly, metal chelate chromatography has more recently assumed a more important role in the purification of proteins. (e.g. U.S. Pat. No. 5,310,663, Döbeli et al, assigned to Hoffman-La Roche Inc.). Simple chelators have been suggested as ligands for IMAC, such as iminodiacetic acid (IDA). IDA, coupled to agarose supports and subsequently charged with various metals, such as Cu2+, Zn2+ and Ni2+, has been used for the capture of proteins and peptides and is also available as a commercial resin. More specifically, U.S. Pat. No. 4,551,271 (Hochuli, assigned to Hoffmann-La Roche Inc.) discloses a metal chelate resin which comprises IDA ligands, in the purification of interferon. The resin can be defined by the following formula:[agarose]-O—(CH2)—CHOH—CH2—N(CH2COO−)2Me2+,wherein Me is Ni or Cu.
The best results are obtained with this resin if the interferon has already been partially purified. The resin can according to the specification be prepared in a known manner by treating agarose with epichlorohydrin or epibromohydrin, reacting the resulting epoxide with iminoacetic acid disodium salt and converting the product into the copper or zinc salt by washing with a copper (II) or zinc solution.
More recently, EP 87109892.7 (F. Hoffmann-La Roche AG) and its equivalent U.S. Pat. No. 4,877,830 (Dobeli et al, assigned to Hoffmann-La Roche Inc.) disclosed a tetradentate chelator known as nitrilotriacetic acid (NTA) for use with metals that have six coordination sites. More specifically, the matrices can be described by the general formula:[carrier matrix]-spacer-NH—(CH2)x—CH(COOH)—N(CH2COO−)2Ni2+,wherein x=2-4.
The disclosed matrix is prepared by reacting an amino acid compound of the formula R—HN—(CH2)x—CH(NH2)—COOH, wherein R is an amino protecting group and x is 2, 3 or 4, with bromoacetic acid in alkaline medium and subsequently, after an intermediate purification step, cleaving off the protecting group and reacting this group with an activated matrix. Accordingly, the method of preparation involves separate steps for alkylating and deprotecting the amino acid, which steps renders the method time-consuming and hence costly. In addition, the alkylation chemistry is less efficient, and after deprotection, the product is not well defined following neutralisation and cleavage. Subsequently, the material is coupled to a solid support that carries carboxyl functionalities by forming an amide bond. However, several problems may be encountered in using this procedure as the media obtained comprises both the desired immobilised chelating ligand as well as some unreacted carboxylic groups and is thus heterogeneous in nature. Furthermore, mono-N-protected amino acid compounds are expensive starting materials, rendering the overall method even more costly.
WO 01/81365 (Sigma-Aldrich Co.) discloses a metal chelating composition that according to the specification is capable of forming relatively stable chelates with metal ions that exhibits an improved selectivity for polyhistidine tagged proteins. According to WO 01/81365, the linkage between the chelator and the resin is an important parameter for the selectivity, and the linkage is a neutral ether, a thioether, a selenoether or an amide. The disclosed compositions are coupled to an insoluble carrier, such as SEPHAROSE™ according to given examples. The chromatographic media is produced in two different ways; either by a solid phase reaction directly on to the pre-activated solid support eventually used in the chromatographic media, or by a separate in-solution synthesis of the intermediate product N,N,N′,N′-tetrakis(carboxymethyl)-L-cystine that is eventually coupled to the solid support.
The solid phase synthesis is carried out by adding L-cysteine to a previously epichlorohydrine activated SEPHAROSE™ gel under alkaline conditions for a prolonged reaction time (18 h), followed by washings. Thereafter bromoacetic acid is added under basic conditions and a prolonged reaction time (72 h), again followed by washings, and any remaining free amino groups present on the gel capped with acetic acid anhydride. Solid phase synthesis in this way offers poor control of the reaction and potential side reactions, and thereby yields a less homogeneous product.
The alternative route, relying on in-solution phase synthesis of an intermediate product starts with addition of a large excess (40 times) of glyoxylic acid to L-cystine in an alkaline borate buffer. The intermediate product, following pH manipulation and conductivity adjustment of the reaction mixture, purified with ion exchange chromatography to give N,N,N′,N′-tetrakis(carboxymethyl)-L-cystine.
Before coupling to a solid support the N,N,N′,N′-tetrakis(carboxymethyl)-L-cystine has to be reduced to N,N-bis(carboxymethyl)-L-cysteine using tris(carboxyethyl)phosphine under alkaline conditions. This material can finally be used for coupling to a pre-activated solid support forming the chromatographic media. This synthetic method is elaborate and depends on a large excess of reagents to form the desired product that is eventually purified under specific chromatographic conditions, followed by reduction as an additional synthetic step, and is thereby less suited for use in large-scale production.
WO 2004/076475 (Andersson et al, Assigned to Amersham Bio-Sciences AB) discloses a method of generating polydentate metal chelating affinity ligands which can subsequently be coupled to a base matrix. The method involves providing a cyclic scaffold comprising a carbonyl, an adjacent sulfphur and a nucleophile; providing a polydentate metal chelating affinity ligand arm on each scaffold by derivatisation of the nucleophile, ring-opening of the cyclic scaffold by addition of reagent that adds more metal chelating affinity ligand arm(s) to the scaffold; and, if required, deprotecting the functionalities of the ligand arm(s). The preferred ligands (which comprise NTA), when coupled to a base matrix, are useful in the purification of his-tagged proteins.
GB707709 discloses the synthesis of pantethein from cysteamine. The compound pantethein does not possess any metal chelating properties.
One key factor in the use of any IMAC ligand in separation media is that of metal binding capacity. The ligand must clearly be able to form a chelate with the transition metal ion of choice for the particular chromatographic separation. The binding capacity of the ligand for the metal ion will influence the conditions required for eluting the adsorbed substance, such as a protein, from the media (for example the pH or concentration of competitor eluant required). Another important factor, which is related to binding capacity, is that of metal leakage from the separation media. Clearly there is a desire to minimise metal leakage from the separation media as metals may be toxic or have adverse effects on the final product of the chromatographic separation. This is particularly true in the field of protein separations where metals may have inhibitory effects on protein function.
Separation media based upon known IMAC ligands vary in the degree to which they chelate or bind metals and also in the extent to which such metals leach from the media on elution with acidic or competitive eluants, such as imidazole.
Accordingly, there is still a need of improved methods for synthesis of IMAC ligands as well as of methods for the immobilisation thereof to a base matrix.